Sensitive Spectrophotometric Assay Research Articles

The effects of hypercholesterolaemia, simvastatin and dietary fat on the low density lipoprotein receptor of unstimulated mononuclear cells

The in vivo expression and regulation of the LDL receptor of circulating mononuclear cells was studied using a sensitive spectrophotometric assay with low density lipoproteins conjugated to colloidal gold (LDL-gold). The high plasma cholesterol of familial hypercholesterolemic subjects was shown to be related to a low in vivo LDL receptor activity; cells from a homozygote had virtually no activity and those from 24 heterozygotes expressed 45% of the activity of cells from 35 normals. The average receptor activity of cells from 18 polygenic hypercholesterolemic (PH) subjects was not significantly different from normal but a low expression may have been a factor in six of these subjects. Simvastatin increased the LDL receptor activity of cells from the PH subjects by 70% while lowering their plasma cholesterol by 26%, but reducing the fat intake from 38% to 20% of energy and cholesterol from 239 to 96 mg/day had no effect on the receptor despite a 10% reduction in plasma cholesterol. Upregulation of the LDL receptor may therefore have been involved in the lowering of plasma cholesterol by simvastatin but not by the reduction in dietary fat and cholesterol.

Spectrophotometric Method for the Assay of Glycosaminoglycans and Glycosaminoglycan-Depolymerizing Enzymes

Undegraded glycosaminoglycans form a complex with 1-ethyl-2-[3-(1-ethylnaphtho-[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naphtho-[1,2-d]thiazolium bromide (Stains-all) in solution resulting in a characteristic shift in spectrum. Hyaluronic acid (a nonsulfated glycosaminoglycan) reacts with the dye to form a complex with an absorbance maximum at 650 nm, while sulfated glycosaminoglycans (chondroitin sulfates A and C, dermatan sulfate, keratan sulfate, and heparan sulfate) give rise to an increase in absorbance at 480 nm. Increases in absorbance at the appropriate wavelength are directly proportional to the concentration of glycosaminoglycan interacting with the dye. This phenomenon provided the basis for a sensitive spectrophotometric assay for the quantitative measurement of bacterial and mammalian glycosaminoglycan-depolymerizing enzymes. The basic assay method was adapted for use in 96-well microtiter trays, thus enabling large numbers of assays to be carried out simultaneously.

An Amplified Assay for Thiols Based on Reactivation of Papain

A sensitive spectrophotometric assay has been developed for thiol (sulfhydryl) groups using an inactive di-sulfide derivative of papain (papain-S-SCH3). The thiol-disulfide interchange reaction of a thiol with papain-S-SCH3 results in the stoichiometric formation of active papain (papain-SH). The reactivated papain catalyzes the hydrolysis of a chromogenic substrate, resulting in an amplified spectrophotometric signal proportional to the initial amount of thiol. A variety of thiols, e.g., cysteine, glutathione, penicillamine, cysteine methyl ester, and cysteamine, yield similar linear plots for the activity of papain vs the initial amount of thiol. An unknown concentration of a thiol is measured using a standard plot for the activity of papain vs the amount of thiol, obtained for the same thiol or for a similar thiol. Thiol groups on proteins and thiol groups of high values of pKa(2-mercaptoethanol, 3-mercaptopropanoic acid) can also be assayed using papain-S-SCH3 in the presence of excess cystamine. The assay is about 100-fold more sensitive than that using Ellman′s reagent [5,5′-dithiobis(2-nitrobenzoic acid)]. A 0.4 μM solution of cysteine produces an absorbance change of 0.55 at 410 nm after 30 mm in the assay, compared to a predicted change in absorbance of 0.0054 using Ellman′s assay.

Chondroitin sulfate depolymerase and hyaluronidase activities of viridans streptococci determined by a sensitive spectrophotometric assay

Sensitive spectrophotometric assays for the detection of bacterial chondroitin sulfate depolymerase and hyaluronidase activities were developed by using Stains-all (1-ethyl-2-[3-(1-ethylnaphtho-[1,2-d]thiazolin-2-ylidene)-2- methylpropenyl]naphtho-[1,2-d]thiazolium bromide). Stains-all interacts with hyaluronic acid to produce a shift in the absorption spectrum with a distinct absorption peak between 620 and 660 nm, while chondroitin sulfate interacts to form a distinct shoulder between 440 and 500 nm. Assays measure undegraded substrate. A collection of 110 strains of viridans streptococci, including representatives of all the currently recognized species, was studied. Streptococcus intermedius and S. constellatus degraded hyaluronic acid, while only strains of S. intermedius, primarily isolated from brain and liver abscesses, produced chondroitin sulfate depolymerase. S. intermedius, of all the viridans streptococci, produces the widest range of glycoprotein- and glycosoaminoglycan-degrading enzymes, which may contribute to the virulence of this species.

Optimized Spectrophotometric Determination of Aldehyde Dehydrogenase Activity in Erythrocytes

We describe a reliable and sensitive semiautomated spectrophotometric assay of aldehyde dehydrogenase (ALDH; EC 1.2.1.3) activity in erythrocytes. The hemolysate can be stabilized with sucrose, and the technique involves only microliters of hemolysate on a centrifugal analyzer. The use of microcolumns to remove interfering hemoglobin is avoided, and reproducibility of the assay has been improved by manipulating the inherent lactate dehydrogenase activity of erythrocytes by adding lactate and oxalate to the reaction mixture. These modifications have decreased the analytical imprecision of the assay, allowing a better appraisal of aldehyde dehydrogenase activity in erythrocytes as a biological marker of excess alcohol consumption. Erythrocytic ALDH activity was significantly less in 40 alcoholics than in 145 teetotallers (median activity 128 vs 219 mU/g of hemoglobin, respectively; P = 0.0001), indicating the potential of this assay as a useful marker of excess alcohol consumption.

A chromogenic substrate for phosphatidylinositol-specific phospholipase C: 4-nitrophenyl myo-inositol-1-phosphate

A chromogenic water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized starting from myo-inositol employing isopropylidene and 4-methoxytetrahydropyranyl protecting groups. In this analogue of phosphatidylinositol, 4-nitrophenol replaces the diacylglycerol moiety, resulting in synthetic, racemic 4-nitrophenyl myo-inositol-1-phosphate. Using this synthetic substrate a rapid, convenient and sensitive spectrophotometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus was developed. Initial rates of the cleavage of the nitrophenol substrate were linear with time and the amount of enzyme used. At pH 7.0, specific activities for the B. cereus enzyme were 77 and 150 μmol substrate cleaved min −1 (mg protein) −1 at substrate concentrations of 1 and 2 mM, respectively. Under these conditions, less than 50 ng quantities of enzyme were easily detected. The chromogenic substrate was stable during long term storage (6 months) as a solid at −20°C.

A sensitive spectrophotometric diamine oxidase activity assay in platelet rich plasma

A sensitive spectrophotometric diamine oxidase activity assay in platelet rich plasma

N,N,N′,N′-Tetramethyl-1,4-phenylenediamine: a facile electron donor and chromophoric substrate for dopamine β-monooxygenase

Dopamine β-monooxygenase is shown to catalyze the oxidation of N,N,N′,N′-tetramethyl-1,4-phenylenediamine (TMPD) to its cation radical in the presence of a regular substrate and molecular oxygen. The enzyme-mediated oxidation of TMPD is stoichiometrically coupled with the hydoxylation of the substrate to the corresponding enzymatic product. TMPD is kinetically well behaved as an alternate electron donor for the enzyme with a potency comparable to that of the most efficient electron donor, ascorbate. Dopamine β-monooxygenase mediated oxidation of TMPD has been employed to design a convenient and sensitive spectrophotometric assay for the enzyme. The finding that TMPD is a well behaved facile alternate electron donor for dopamine β-monooxygenase raises some interesting novel questions regarding the specificity and chemistry of the reduction site, which may have important implications on the reduction of active site coppers of the enzyme.

A spectrophotometric assay for quantitative analysis of the oxidation of 4-hydroxystilbene by peroxidase-H 2O 2 systems

A new and sensitive spectrophotometric assay has been developed to study and to characterize kinetically the oxidation of 4-hydroxystilbene (an analogue of the putative viniferin precursor, trans-resveratrol, directly involved in the resistance mechanism of the grapevine to fungal diseases) by peroxidase-H 2O 2 systems. The technique measures the overall increase in absorbance at 248 nm in the reaction media, probably due to the formation of a phenoxy radical of the 4-hrdroxystilbene (4-HS). This technique was developed by using the purified isoenzyme C of horseradish peroxidase and all the validity criteria (sensitivity and reproducibility) were checked. The results show that it is especially suitable for low activity measurements. It was finally applied to the determknation of the oxidation rate of 4-HS by peroxidases isolated from the media of suspension-cultured grapevine cells, at two different developmental stages.

The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII

In the extrinsic pathway of blood coagulation, Factor X is activated by a complex of tissue factor, factor VII(a) and Ca2+ ions. Using purified human coagulation factors and a sensitive spectrophotometric assay for Factor Xa, we could demonstrate activation of Factor X by Factor VIIa in the absence of tissue-factor apoprotein, phospholipids and Ca2+. This finding allowed a kinetic analysis of the contribution of each of the cofactors. Ca2+ stimulated the reaction rate 10-fold at an optimum of 6 mM (Vmax. of 1.1 x 10(-3) min-1) mainly by decreasing the Km of Factor X (to 11.4 microM). In the presence of Ca2+, 25 microM-phospholipid caused a 150-fold decrease of the apparent Km and a 2-fold increase of the apparent Vmax. of the reaction; however, both kinetic parameters increased with increasing phospholipid concentration. Tissue-factor apoprotein contributed to the reaction rate mainly by an increase of the Vmax., in both the presence (40,500-fold) and absence (4900-fold) of phospholipid. The formation of a ternary complex of Factor VIIa with tissue-factor apoprotein and phospholipid was responsible for a 15 million-fold increase in the catalytic efficiency of Factor X activation. The presence of Ca2+ was absolutely required for the stimulatory effects of phospholipid and apoprotein. The data fit a general model in which the Ca2(+)-dependent conformation allows Factor VIIa to bind tissue-factor apoprotein and/or a negatively charged phospholipid surface resulting into a decreased intrinsic Km and an increased Vmax. for the activation of fluid-phase Factor X.

Solid-supported single- and multi-enzyme amplified assays for clostridiopeptidase A

A rapid and sensitive spectrophotometric assay for Clostridium histolyticum clostridiopeptidase A (collagenase) was accomplished by measuring the activity of an alkaline phosphatase indicator enzyme released into solution from insoluble, covalently linked alkaline phosphatase indicator enzyme released into solution from activity of the alkaline phosphatase was monitored spectrophotometrically using either p-nitrophenyl phosphate as substrate or more sensitively by a signal amplification system consisting of NAD +, alcohol dehydrogenase, diaphorase and INT-Violet. Under the reaction conditions the amount of indicator enzyme produced is directly proportional to the concentration of collagenase. With p-nitrophenyl phosphate as substrate the magnitude of the signal was 0.003 abs. min −1 per 100 ng ml −1 collagenase whereas with the multienzyme amplification system it was 0.035 abs. min −1, i.e. approximately as 12-fold increase. The method consists in first incubating the substrate with the bacterial collagenase for 20 min, then up to 96 samples of the released alkaline phosphatase can be analysed in 2 min using a microtitre plate reader run in the kinetic mode.

3,4-Dinitrophenyl N-acetyl-β- d-glucosaminide, a synthetic substrate for direct spectrophotometric assay of N-acetyl-β- d-glucosaminidase or N-acetyl-β- d-hexosaminidase

3,4-Dinitrophenyl N-acetyl-β- d-glucosaminide (3,4-dnpGlcNAc) was synthesized and proposed as an artificial substrate for N-acetyl-β- d-glucosaminidase (EC 3.2.1.30) and N-acetyl-β- d-hexosaminidase (EC 3.2.1.52) (collectively abbreviated as NAGase). 3,4-dnpGlcNAc is water soluble and is fairly stable in aqueous medium. 3,4-Dinitrophenol released from 3,4-dnpGlcNAc by NAGase absorbs light of 400 nm at pH near 5, where the activity of urinary NAGase is assayed for diagnostic use. Direct and sensitive spectrophotometric assay of NAGase is, thus, possible using 3,4-dnpGlcNAc as an artificial substrate by monitoring the increase of A 400 due to the release of 3,4-dinitrophenol in a thermostated spectrophotometer.

A simple procedure using trinitrobenzenesulphonic acid for monitoring proteolysis in cheese

SummaryA simple, rapid and sensitive spectrophotometric assay was developed and evaluated for monitoring proteolysis during cheese ripening, based on the fact that α-amino groups released by hydrolysis of cheese proteins react with trinitrobenzenesulphonic acid to form products that absorb strongly at 420 nm. A linear relationship was shown to exist between A420 and concentration of free α amino groups up to 0·5 HIM (r = 0·999, 38 df, P < 0·001). Repeatability of the method was satisfactory. The coefficient of variance was 0·53% for amino acid solutions and 1·19% for cheese extracts. Average recovery of glycine added to the cheese was 104 ± 2·9%. A comparison of the above method with that of determination of water-soluble N to total N ratio showed that there was good agreement between these two methods of assessment of proteolysis in cheese (r = 0·857, 32 df, P < 0·001). Mainly Feta and Teleme cheese were examined, but a similar correlation was obtained with hard Greek cheeses. Analytical conditions of the procedure are discussed.

A rapid and simple MTT-based spectrophotometric assay for determining drug sensitivity in monolayer cultures

A rapid and sensitive spectrophotometric assay for determining viability in monolayer cultured cell lines, with specific applications in normal and drug resistant cell line determinations, is described. The assay involves conversion of the tetrazolium salt MTT by viable proliferating cells to an insoluble product, purple formazan. The chief advantage of this assay is that it requires fewer cells than standard cytotoxicity assays. In addition, it allows for multiple sample concentrations on a single 96-well plate which is then rapidly quantitated using an automated spectrophotometric microplate reader.

A simple spectrophotometric assay for micromolar amounts of lanthanum in the presence of calcium and phosphate.

A sensitive spectrophotometric assay for micromolar amounts of lanthanum in the presence of calcium and phosphate (as hydroxyapatite) was developed utilizing the change in absorption (at 652 nm) when the dye arsenazo III was complexed with lanthanum. Arsenazo III was used at a level of 25 microM and the solution pH was maintained at 3.1 with 0.2 M sodium acetate. Lanthanum concentrations down to 0.5 microM could be reliably assayed. Calcium ion did not complex well with arsenazo III at pH 3.1. With calcium present at 100 microM and lanthanum at 10 microM, the assay was 115 times more sensitive for lanthanum. The assay is simple, rapid, reproducible and, unlike the assay using radioactive lanthanum, can be performed at any time.

Spectrophotometric assay for ornithine decarboxylase

A rapid and sensitive spectrophotometric assay for ornithine decarboxylase is described. It is based on the observation that the product of ornithine decarboxylase, putrescine, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a colored product soluble in 1-pentanol whereas ornithine does not. The amount of putrescine produced by the enzyme was determined by measuring the absorbance of the 1-pentanol extract of the reaction mixture at 420 nm, and by comparing the results to those obtained by the trapping of 14CO 2 and by HPLC assays. The three assays were found to be equivalent in sensitivity, with the spectrophotometric assay having the advantages of being relatively rapid, requiring only common laboratory equipment, and not requiring the use of radioactive isotopes.

A sensitive spectrophotometric assay of pyruvate dehydrogenase activity

The recently reported highly sensitive method for assay of acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) [ H. H. Andres, A. J. Klein, S. M. Szabo, and W. W. Weber (1985) 145, 367–375] has been adapted for determination of pyruvate dehydrogenase activity. This method provides an improvement in sensitivity over extant spectrophotometric methods and circumvents limitations of assays using radioactive pyruvate. In addition, the assay is simple and inexpensive and can be readily adapted for measurement of enzyme activity in crude tissue extracts or homogenates.

Cytochrome P-450 activity in human leiomyoma and normal myometrium

Variations in cytochrome P-450 levels may influence the responsiveness of uterine and breast tissue as well as carcinomas to endocrine therapy and may be of particular importance with agents such as tamoxifen (Nolvadex) where hydroxylation is known to alter therapeutic activities. Therefore, a sensitive spectrophotometric assay of cytochrome P-450 levels in reproductive tissue microsomes was developed to measure cyclohexane hydroxylase activity. Cyclohexane served as a substrate for several forms of cytochrome P-450. Human uterine leiomyomas (uterine fibroid tumor) contained significantly higher (p < 0.01) cytochrome P-450 activity than adjacent normal myometrium. Specific activities for both leiomyomas (2.87 ± 0.26 nmol/min/mg) and normal myometrium (1.60 ± 0.11 nmol/min/mg) were in the range of those observed for untreated rabbit liver microsomes (1 to 3 nmol/min/mg). The contribution of smooth muscle in the specimen, the phase of the menstrual cycle, and the clinical diagnosis did not influence the level of cytochrome P-450 activity.

Quantification of cytochrome P-450-dependent cyclohexane hydroxylase activity in normal and neoplastic reproductive tissues.

It is well established that liver microsomal cytochrome P-450 participates in steroid metabolism and probably also in the metabolism of anti-oestrogens such as tamoxifen (Nolvadex). Thus it is possible that variations in cytochrome P-450 levels may influence the responsiveness of human breast and endometrial carcinomas to endocrine therapy. Therefore a simple sensitive spectrophotometric assay for determining levels of cytochrome P-450-dependent cyclohexane hydroxylation activity in breast and uterine microsomes (microsomal fractions) has been developed. Cyclohexane was chosen as a substrate because of the relatively high levels of cyclohexane hydroxylase activity in tumour microsomes and because cyclohexane serves as a substrate for several forms of cytochrome P-450. As previously described [Senler, Dean, Pierce & Wittliff (1985) Anal. Biochem. 144, 152-158], a direct method utilizing isotope-dilution/gas chromatography-mass spectrometry was also developed in order to confirm the results of the spectrophotometric assay. The average activity (cyclohexane-dependent NADPH oxidation) for 139 human breast-tumour microsome preparations was 1.34 nmol/min per mg, which is in the range of that found in untreated mammalian liver (1-3 nmol/min per mg). Also, high enzyme activity was demonstrated in human ovary, normal uterus as well as uterine leiomyomas. Endocrine status appeared to influence enzyme levels, in that mammary tissue from virgin rats contained significantly (P less than 0.025) higher amounts of activity than did tissues from either pregnant or lactating rats. Furthermore, carbon monoxide, as well as an antibody against rat liver cytochrome P-450, completely inhibited NADPH oxidation by breast-carcinoma microsomes. These results strengthen our hypothesis that tumours with high levels of cytochrome P-450 may have a reduced response to additive endocrine therapy.

Procedures for measuring cytochrome P-450-dependent hydroxylation activity in reproductive tissues

Cytochrome P-450 is present in the endoplasmic reticulum at varying concentrations in almost all tissues. However, the existence and role of cytochrome P-450 in normal and neoplastic reproductive tissues has not been clearly demonstrated. Our interest lies in the possibility that variations in cytochrome P-450 levels may influence the responsiveness of breast and endometrial carcinomas to endocrine therapy. This may be of particular importance with agents such as tamoxifen where hydroxylation reactions are known to alter therapeutic activities. Therefore, a simple, sensitive spectrophotometric assay for determining levels of cytochrome P-450-dependent cyclohexane hydroxylase activity in breast and uterine microsomes has been developed. Cyclohexane was chosen as a substrate because of the relatively high levels of cyclohexane hydroxylase activity in tumor microsomes and because cyclohexane serves as a substrate for several forms of cytochrome P-450. In order to confirm the results of the spectrophotometric assay, a direct method utilizing isotope dilution gas chromatography/mass spectrometry ( GC MS ) has been developed for detecting low levels of the hydroxylated product, cyclohexanol. By employing a stable isotopically labeled analog of cyclohexanol (cyclohexanol-d 12), good agreement was demonstrated between the simple, indirect method (measuring NADPH oxidation at 340 nm) and the more complex, direct method (measuring cyclohexanol formation) utilizing GC MS . The agreement of results obtained using these two techniques indicates that they are equally valid measures of NADPH-dependent cyclohexane hydroxylase activity. The use of the spectrophotometric method is proposed for rapid, multiple assays such as in the clinical setting, reserving GC MS analysis for use as a research tool.